All living creatures including humans are made of cells. The majority of life forms exist as single cells that perform all functions to continue independent life. A number of cells, cell organelles, bacteria and viruses are too small to be seen with traditional optical microscopes. To view cellular life, the microscope must have high resolution.
Resolution is the ability of a microscope to distinguish between two objects that are very close together. A microscope with a resolution of 1,000 Å (1,000 Angstroms; equal to 100 nanometers), for example, can make objects as close together as 100 nanometers independently visible. Objects and features smaller than 100 nanometers cannot be resolved (i.e., distinguished) by a microscope with a resolution of 1000 Å.
Currently there are a few microscopes which offer very fine resolution. One example is the “High-Resolution Optical Microscope” described in U.S. Pat. No. 6,690,509.
Electron microscopes are another example. However, in using electron microscopes, the specimen must be prepared by high-vacuum dehydration and is subjected to intense heat by the electron beam, making observation of living specimens impossible. The sample preparation for electron microscopy requires living cells to be killed, frozen, dehydrated and impregnated with heavy metals. The dehydration process also alters the specimen, leaving artifacts and cell damage that were not present in nature. These procedures, together with electron beam damage caused during the observation of the cells creates the possibility that some components of the cell may be lost or destroyed. Also, in order to view the steps in a biological process, dozens of specimens must be viewed at various stages in order to capture each desired step. Further, specimen preparation for electron microscopes can take up to two hours each.
The high cost of an electron microscope represents another barrier to its use in the life sciences. Electron microscopes are large and often require an entire room. The operation and adjustment of an electronic microscope also requires highly skilled technicians.
The ultraviolet microscope offers finer resolution and better magnification than an ordinary light microscope, but it has serious disadvantages for the study of living specimens. Ultraviolet light damages or kills many kinds of living biological specimens, making observation impossible. In ultraviolet microscopy specimens are often stained with a fluorescent dye. Many fluorescent dyes bind strongly to elements such as enzymes within living cells, changing their qualities and significantly altering the cellular biochemistry. Other dyes produce too much fluorescence or absorb too much of the ultraviolet light to be useful.
Like electron microscopes, the operation of an ultraviolet microscope requires a great deal of skill. Because ultraviolet light damages the human eye, the image can only be observed by ultraviolet video cameras or specially-equipped still cameras. Also, the quartz optics required for ultraviolet microscopes are much more expensive than the glass components used in visible light microscopes.
The electron and ultraviolet microscopes available today do not offer a technique for observing living, unaltered biological specimens in real time. The damage from the electronic beam, from fixing, freezing, exposure to ultraviolet light, and other procedures which occur during observation limits the processes which can be seen by such devices. For example, the phenomenon of cellular transport cannot be observed in non-living cells by these known devices. Other examples of phenomena which cannot be directly observed by electronic microscopes include: streaming, Brownian motion, diffusion, phagocytosis, pinocytosis, mitosis, immuno-fluorescence, and cell interactions.
It is important for scientists in the biomedical community to observe these living cells and their processes in order to better understand the cellular world. Currently, the behavior cells and the phenomena responsible can only be inferred. These processes can only be studied in depth while they are occurring and while the cells are alive. Observing live cellular activity is needed to more completely understand such processes as gene therapy, artificial insemination, new drug development, cell culturing and cloning, cell regeneration, implantation, bio-detection, and biotherapeutics, amongst others. Observing very small, live cells at a high resolution, with a high contrast could possibly lead to the development of treatments to diseases and other health problems.